Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. (2011) Treating Cancer with Genetically Engineered T Cells. Trends Biotechnol. 29(11): 550-557) Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma.
Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. (2010) Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor. Blood. 116(7): 1035-1044). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains to form a single-chain fusion molecule. However, this approach has so far proven efficiency only with respect to patients with acute lymphoblastic leukemia (ALL) by targeting malignant B cells bearing the antigen CD19 (Porter, D. L. et al. (2011) Chimeric Antigen Receptor—Modified T Cells in Chronic Lymphoid Leukemia. N. Engl. J. Med. 365:725-733). Chronic lymphocytic leukemia (CLL) is one of the most commonly diagnosed leukemias managed by practicing hematologists. For many years patients with CLL have been viewed as similar, with a long natural history and only marginally effective therapies that rarely yielded complete responses. Recently, several important observations related to the biologic significance of VH mutational status and associated ZAP-70 overexpression, disrupted p53 function, and chromosomal aberrations have led to the ability to identify patients at high risk for early disease progression and inferior survival. Concurrent with these investigations, several treatments including the nucleoside analogues, monoclonal antibodies rituximab and alemtuzumab have been introduced. Combination of these therapies in clinical trials has led to high complete and overall response rates when applied as initial therapy for symptomatic CLL. Thus, the complexity of initial risk stratification of CLL and treatment has increased significantly. Furthermore, when these initial therapies do not work, approach of the CLL patient with fludarabine-refractory disease can be quite challenging (Byrd J. C et al, 2014).
One candidate antigen of immunotherapies for chronic lymphocytic leukemia (CLL) is Tyrosine-protein kinase transmembrane receptor ROR1 (also called NTRKR1; UniProtKB/TrEMBL) entries: Q01973). ROR1 (The receptor tyrosine kinase-like orphan receptor 1) is a 120-kDa glycoprotein containing an extracellular immunoglobulin (Ig)-like, Kringle, and Frizzled-like cysteine rich domain (FIG. 1). The protein encoded by this gene is a receptor tyrosine kinase that modulates neurite growth in the central nervous system. It is a type I membrane protein and belongs to the ROR subfamily of cell surface receptors (Reddy et al, 1997). The Ror1 protein expression in patients with CLL but not in normal leukocytes merits further studies of its role in the pathobiology of CLL, which may provide a basis for development of Ror1 directed targeted therapy (Daneshmanesh et al; 2008). ROR1 is expressed on a variety of B-cell malignancies, and subsets of some solid tumors, including breast, colon, lung, and kidney tumors. ROR1 functions in oncogenic signaling to promote tumor cell survival in epithelial tumors. Importantly, ROR1 is not expressed on vital organs, except adipose and pancreatic tissue, which reduces potential toxicities from killing of normal cells (Hudecek et al, 2013). ROR1 is expressed during embryogenesis but absent from normal adult tissues, apart from a subset of immature B-cell precursors, and low-level expression on adipocytes (Hudecek et al., 2010; Matsuda et al., 2001). ROR1 was first shown to be expressed in B-cell chronic lymphocytic leukemia (B-CLL) by transcriptional profiling (Klein et al., 2001; Rosenwald et al., 2001) and was subsequently identified on the surface of many cancers including mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL) with a t(1;19) chromosome translocation, and a subset of lung, breast, colon, pancreas, renal, and ovarian cancers (Baskar et al., 2008; Bicocca et al., 2012; Daneshmanesh et al., 2008; Dave et al., 2012; Fukuda et al., 2008; Yamaguchi et al., 2012; Zhang et al., 2012a, 2012b). In both lung adenocarcinoma and t(1;19) ALL, ROR1 cooperates in oncogenic signaling and knockdown of ROR1 with siRNA exposed a critical role for this molecule in maintaining tumor cell survival (Bicocca et al., 2012; Choudhury et al., 2010; Gentile et al., 2011; Yamaguchi et al., 2012). Thus, ROR1 loss may not be readily tolerated by tumors making it an attractive candidate for CAR directed T-cell therapy that could be broadly applied. It thus represents an appropriate target antigen for treating CLL or solid tumors, especially using CAR-expressing T cells.
The laboratories of Dr. Stanley Riddell and Dr. Laurence Cooper have previously engineered and validated anti-ROR1 scCARs containing the 4A5 and the 2A2 scFvs, respectively (Cooper et al 2010; Hudecek et al., 2013). In particular, Hudecek et al discloses anti-ROR1 scCARs which contain an IgG4 hinge of diverse length and a CD28 transmembrane domain.
There is still the need for the improvement of CAR functionality by designing CAR architecture and using suitable components since these parameters play a role important and a fine tuning is necessary.
In the context of developing therapeutic grade engineered immune cells that can target malignant or infected cells, the inventors have sought for improved CAR architectures, which would be closer to natural ones and likely to behave accordingly using any extracellular mono or multi-specific ligand binding domains. In WO2014039523, they described a new generation of CARs involving separate polypeptide sub-units according to the present invention, referred to as “multi-chain CARs”. According to this architecture, the signaling domains and co-stimulatory domains are located on different polypeptide chains (FIG. 2). Such multi-chain CARs can be derived from FcεRI, by replacing the high affinity IgE binding domain of FcεRI alpha chain by an extracellular ligand-binding domain such as scFv, whereas the N and/or C-termini tails of FcεRI beta and/or gamma chains are fused to signal transducing domains and co-stimulatory domains respectively. The extracellular ligand binding domain has the role of redirecting T-cell specificity towards cell targets, while the signal transducing domains activate the immune cell response. The fact that the different polypeptides derived from the alpha, beta and gamma polypeptides from FcεRI are transmembrane polypeptides sitting in juxtamembrane position, provides a more flexible architecture to CARs and reduces background activation of immune cells. However, this flexibility provides more variability from one binding sequence to another, so that it is difficult to predict which binding domain and optimal architecture provide with an appropriate specificity towards ROR1.
It can be noted that single and multichain CAR architectures bearing the same scFvs may not perform the same way, depending of parameters which are not always controlled by the skilled man of the art. This remark may apply also to the type of expression used (transient or stable by using respectively, for instance, mRNA or lentivirus delivery).
Another aspect to be considered is the potential adverse effects linked to the infusion of engineered T cells to the patient, and in particular the cytokine-release syndrome (CRS). Thus, there is the need for designing the right CAR architecture and their specific components which can reduce the occurrence of such adverse events.
The invention provides with optimally designed multi-chain CAR bearing scFv extracellular domain, which are particularly suited to target malignant cells bearing ROR1 as a surface protein. It has been shown in the present invention that a particular architecture of multichain CAR with well-defined components can allow the engineered immune cells to be cytotoxic towards ROR1 antigen-bearing tumor cells. From those mcCARs, 2 of them csm13 and csm14 appear to be performant in terms of specific lysis while the immune cells keep their innate function.
This achievement opens the way to new immunotherapy treatments of malignant cells diagnosed to be ROR1 positive, such as those found in CLL and solid tumors in particular breast, colon, lung, and kidney tumors.